Porto Conte RicerchePorto Conte Ricerchehttp://hdl.handle.net/11050/32018-05-24T17:52:14Z2018-05-24T17:52:14ZOral contraceptives modify DNA methylation and monocyte-derived macrophage functionCampesi, IlariaSanna, ManuelaZinellu, AngeloCarru, CiriacoRubattu, LauraBulzomi, PamelaSeghieri, GiuseppeTonolo, GiancarloPalermo, MarioRosano, GiuseppeMarino, MariaFranconi, Flaviahttp://hdl.handle.net/11050/11452015-01-23T07:49:26Z2012-01-27T00:00:00ZOral contraceptives modify DNA methylation and monocyte-derived macrophage function
Campesi, Ilaria; Sanna, Manuela; Zinellu, Angelo; Carru, Ciriaco; Rubattu, Laura; Bulzomi, Pamela; Seghieri, Giuseppe; Tonolo, Giancarlo; Palermo, Mario; Rosano, Giuseppe; Marino, Maria; Franconi, Flavia
Background: Fertile women may be encouraged to use contraception during clinical trials to avoid potential drug effects on fetuses. However, hormonal contraception interferes with pharmacokinetics and pharmacodynamics and modifies internal milieus. Macrophages depend on the milieu to which they are exposed. Therefore, we assessed whether macrophage function would be affected by the use of combined oral contraceptives (OCs) and if this influence depended on the androgenic or non-androgenic properties of progestin.
Methods: Healthy adult women were enrolled and stratified into two groups: women who did not use OCs (Fs) and women treated with OCs (FOCs). FOCs were further stratified as a function of androgenic (FOCA+) and non-androgenic (FOCA-) properties of progestins. Routine hematological, biochemical, inflammatory and endothelial dysfunction parameters were measured. Monocyte-derived macrophages (MDMs) were evaluated for the expression and activity of estrogen receptors and androgen receptors, and release of tumor necrosis factor α (TNFα) was measured from unstimulated and lipopolysaccharide-stimulated cells.
Results: As is already known, the use of OCs changed numerous parameters: the number of lymphocytes, iron levels, total iron-binding capacity of transferrin, triglycerides, high-density lipoprotein, total cholesterol, and C-reactive protein increased, while prothrombin time and alkaline phosphatase decreased. Hormonal levels also varied: cortisol was higher in FOCs, while luteinizing hormone, follicle-stimulating hormone, and testosterone were lower in FOCs. Asymmetric dimethylarginine, an index of endothelial function, was lower in FOC than in Fs, as were cysteine and bilirubin. The androgenic properties of progestins affected the activity of OCs: in particular, white blood cell count, hemoglobin, high-density lipoprotein and calcium were higher in FOCA- than in FOCA+, whereas percentage oxygen saturation and γ-glutamyl transpeptidase were lower in FOCA- than in FOCA+. Importantly, FOCs had a lower global DNA methylation, indicating that OC may have epigenetic effects on gene expression. OC did not modify the expression of androgen receptor but increased estrogen receptor α expression, more considerably in FOCA+, and decreased estrogen receptor β, more considerably in FOCA-. Importantly, the activation state of estrogen receptor β in FOCs was decreased, while estrogen receptor α was not active in either Fs or FOCs. Unstimulated MDMs obtained from FOCs showed higher release of TNFα in comparison with Fs. After lipopolysaccharide stimulation, the release of TNFα was significantly higher in Fs than in FOCs.
Conclusions: OC use induced many changes in hematological and plasmatic markers, modifying hormonal levels, endothelial function, inflammation index and some redox state parameters, producing a perturbation of the internal milieu that impacted macrophagic function. In fact, different levels of estrogen receptor expression and release of TNFα were observed in macrophages derived from OC users. Some of the above activities were linked to the androgenic properties of progestin. Even though it is not known whether these effects are reversible, the results indicate that to avoid potential skewing of results only a single type of OC should be used during a single clinical trial.
2012-01-27T00:00:00ZMolecular changes induced by the curcumin analogue D6 in human melanoma cellsRozzo, CarlaFanciulli, ManuelaFraumene, CristinaCorrias, AntonioCubeddu, TizianaSassu, IlariaCossu, SaraNieddu, ValentinaGalleri, GraziaAzara, EmanuelaDettori, Maria AntoniettaFabbri, DavidePalmieri, GiuseppePisano, Marinahttp://hdl.handle.net/11050/11442015-01-23T07:49:25Z2013-05-04T00:00:00ZMolecular changes induced by the curcumin analogue D6 in human melanoma cells
Rozzo, Carla; Fanciulli, Manuela; Fraumene, Cristina; Corrias, Antonio; Cubeddu, Tiziana; Sassu, Ilaria; Cossu, Sara; Nieddu, Valentina; Galleri, Grazia; Azara, Emanuela; Dettori, Maria Antonietta; Fabbri, Davide; Palmieri, Giuseppe; Pisano, Marina
Background: In a previous report, we described the in vitro and in vivo antiproliferative and proapoptotic activity of a hydroxylated biphenyl (D6), a structural analogue of curcumin, on malignant melanoma and neuroblastoma tumours. In this paper, we investigated the molecular changes induced by such a compound, underlying cell growth arrest and apoptosis in melanoma cells.
Results: To shed light on the mechanisms of action of D6, we firstly demonstrated its quick cellular uptake and subsequent block of cell cycle in G2/M phase transition. A gene expression profile analysis of D6-treated melanoma cells and fibroblasts was then carried out on high density microarrays, to assess gene expression changes induced by this compound. The expression profile study evidenced both an induction of stress response pathways and a modulation of cell growth regulation mechanisms. In particular, our data suggest that the antiproliferative and proapoptotic activities of D6 in melanoma could be partially driven by up-regulation of the p53 signalling pathways as well as by down-regulation of the PI3K/Akt and NF-kB pathways. Modulation of gene expression due to D6 treatment was verified by western blot analysis for single proteins of interest, confirming the results from the gene expression profile analysis.
Conclusions: Our findings contribute to the understanding of the mechanisms of action of D6, through a
comprehensive description of the molecular changes induced by this compound at the gene expression level, in agreement with the previously reported anti-tumour effects on melanoma cells.
2013-05-04T00:00:00ZA straightforward and efficient analytical pipeline for metaproteome characterizationTanca, AlessandroPalomba, AntonioPisanu, SalvatoreDeligios, MassimoFraumene, CristinaManghina, ValeriaPagnozzi, DanielaAddis, Maria FilippaUzzau, Sergiohttp://hdl.handle.net/11050/11432015-01-22T11:11:22Z2014-12-10T00:00:00ZA straightforward and efficient analytical pipeline for metaproteome characterization
Tanca, Alessandro; Palomba, Antonio; Pisanu, Salvatore; Deligios, Massimo; Fraumene, Cristina; Manghina, Valeria; Pagnozzi, Daniela; Addis, Maria Filippa; Uzzau, Sergio
Background: The massive characterization of host-associated and environmental microbial communities has represented a real breakthrough in the life sciences in the last years. In this context, metaproteomics specifically enables the transition from assessing the genomic potential to actually measuring the functional expression of a microbiome. However, significant research efforts are still required to develop analysis pipelines optimized for metaproteome characterization.
Results: This work presents an efficient analytical pipeline for shotgun metaproteomic analysis, combining
bead-beating/freeze-thawing for protein extraction, filter-aided sample preparation for cleanup and digestion, and single-run liquid chromatography-tandem mass spectrometry for peptide separation and identification. The overall procedure is more time-effective and less labor-intensive when compared to state-of-the-art metaproteomic techniques. The pipeline was first evaluated using mock microbial mixtures containing different types of bacteria and yeasts, enabling the identification of up to over 15,000 non-redundant peptide sequences per run with a linear dynamic range from 104 to 108 colony-forming units. The pipeline was then applied to the mouse fecal metaproteome, leading to the overall identification of over 13,000 non-redundant microbial peptides with a false discovery rate of <1%, belonging to over 600 different microbial species and 250 functionally relevant protein families. An extensive mapping of the main microbial metabolic pathways actively functioning in the gut microbiome was also achieved.
Conclusions: The analytical pipeline presented here may be successfully used for the in-depth and time-effective characterization of complex microbial communities, such as the gut microbiome, and represents a useful tool for the microbiome research community.
2014-12-10T00:00:00ZMycoplasma agalactiae MAG_5040 is a Mg2+ -Dependent, Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural InfectionCacciotto, CarlaAddis, Maria FilippaCoradduzza, ElisabettaCarcangiu, LauraNuvoli, Anna MariaTore, GessicaDore, Gian MarioPagnozzi, DanielaUzzau, SergioChessa, BernardoPittau, MarcoAlberti, Albertohttp://hdl.handle.net/11050/11412014-12-18T08:40:08Z2013-02-28T00:00:00ZMycoplasma agalactiae MAG_5040 is a Mg2+ -Dependent, Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection
Cacciotto, Carla; Addis, Maria Filippa; Coradduzza, Elisabetta; Carcangiu, Laura; Nuvoli, Anna Maria; Tore, Gessica; Dore, Gian Mario; Pagnozzi, Daniela; Uzzau, Sergio; Chessa, Bernardo; Pittau, Marco; Alberti, Alberto
In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.
2013-02-28T00:00:00Z